DE2552510C3 - Biologically active compounds and processes for their preparation - Google Patents

Description

The invention relates to new compounds of one Copolymers of vinyl glycol and a biologically active substance chemically bound to it, Process for their preparation and their use, preferably in affinity chromatography.

In the past few years, a new technique has emerged within the more biochemical working methods and more enforced, whose primary characteristic is the affinity of carrier-bound, biological to use active substances for selective reactions.

Via a specific complex formation of the carrier-bound substance with a second, which in one If a mixture is present, the substance in question can be removed from the mixture and, if desired, then isolated by desorption.

Carrier-bound enzymes offer the advantage of carrying out material conversions in preparative, continuous processes and the reaction products to get enzyme-free.

Are in the biochemical, enzymatic analysis Carrier-bound, water-insoluble enzymes are available as re-usable reagents.

Due to the property of the enzymes, affinities not only towards the substrates, but also to exhibit in relation to the specific inhibitors, the extraction of enzyme inhibitors with the help has to be found affinity chromatography on carrier-bound enzymes has proven particularly beneficial. on the other hand the binding of inhibitors to water-insoluble matrices allows the preparative production of the corresponding enzymes.

Antigens or antibodies on water-insoluble matrices are used as so-called immunoadsorbents bound and then allow the isolation of the corresponding antibodies or antigens.

As biologically active substances according to the preceding statements in vivo and in Vitro effective natural and artificially produced substances understood in the broadest sense as enzymes. Activators. Inhibitors, antigens or antibodies. Vitamins and hormones can be designated. For These biologically active substances have been called effectors because they represent the active principles of the water-insoluble systems.

Most of the carrier-bound effectors described so far are much more stable than the corresponding biologically active substances in solution.

As carrier materials, so-called matrices, are advantageous to use those substances that in addition to Insolubility in aqueous systems have the lowest possible unspecific adsorption. In addition must have hydrophobic, hydrophilic and ionic interactions between the template and the reaction * partner of the effector are largely prevented. With substances whose binding to the effector is undesirable. z. B. those who are not specific reaction partners of the effector, a bond should be excluded.

The matrices used so far as carriers for biologically active substances can be subdivided in those who bind the effectors through physical adsorption, for example polystyrene, activated carbon and glass beads and those who are linked with the effectors via a covalent bond. The latter include vinyl polymers as homo- and copolymers, ζ. Β.

Polyacrylic acids, polyacrylic acid amides and amino, Carboxy- or sulfonyl-substituted polystyrene, furthermore cellulose and its derivatives and finally natural and synthetic polypeptides and proteins a. Because of the balanced interaction between the die and the effector, the use of Carbohydrates, especially cellulose, dextran, starch, agar or their derivatives, the highest as a template in aqueous systems

ίο found widespread use, albeit in these Carboxyl groups often contained in natural substances are disruptive because of their unspecific affinities be felt. In addition, these substances have a relatively low thermal and chemical stability.

is Many of these disadvantages could be solved by using of polyvinyl glycol, a synthetic polymer, can be largely eliminated as a matrix (DE-OS 24 21 789). Polyvinyl glycol, also polyhydroxymethylene called, is characterized by acidic or basic hydrolysis

μ polyvinylene carbonate produced every carbon atom carries a hydroxyl group and a hydrogen atom. The continuous -C-C bond gives the Carrier a particularly high stability

Polyvinylene carbonate and copolymers of vinyl

Carbonates have also already been used as carriers for various effectors, in particular proteins (DE-OS 24 07 340). These well-known However, compounds have - if at all - active groups of the vinylene carbonate mono.nereneinheit

jo are not tied to the effector - intact Carbonate groups that do not give the carrier-effector system any hydrophilic properties.

It has now been found that, instead of polyvinyl glycol, it is advantageous to use vinyl glycol copolymers as

α carrier matrix can use. By polymerizing suitable comonomers into polyvinylene carbonate, the physical and chemical properties of the polyvinyl glycol produced therefrom can be varied to a large extent.

So z. B. the "hydrophilicity". or "swelling" of the Polyvinyl glycol by polymerizing in hydrophobic monomers such as ethylene. Vinyl chloride or Styrene can be changed into the polyvinylene carbonate. The copolymerization of vinyl acetate in the saponified end product replaced a vinyl glycol group with a vinyl alcohol group, which increased Swellability in an aqueous medium has the consequence.

It is possible with the help of comonomers, such as. B. Diethylene glycol divinyl ether and divinylbenzene ones

V) networking and thus an improvement in mecham see properties of the carrier obtained by polymerizing electrophilic functions. / B Oxira groups (epoxy groups) from vinyl glycidyl ether comonomers. is a direct implementation of the

«Matrix with nucleophilic functions (-NHj. -OH etc.) possible. Carboxyl groups can be introduced through acrylic acid ester comonomers. After him Following copolymerization, the ester groups and carboxyl groups are saponified. The introduction of -NH?

or -COOH-G <-jppen allows activation with Help from Carbgdiimjden, IsQsasoliumsateen, Gluiar aldehyde etc. Copolymers vinyl glycol / vinyl alcohol have a higher specific surface and damii eim higher binding capacity for e.g. B. Proteins at the

to surface of the polymer powder compared to vinyl glycol homopolymer

The invention accordingly relates to compounds made from a copolymer of vinyl glycol and

one of them while maintaining their biological activity chemically bound biologically active substance, in which only part of the active groups of the vinyl glycol units of the polymer to the biologically active Substance is bound and the remainder is present as free hydroxyl groups

In these compounds, the carrier matrix is a polyvinyl glycol copolymer containing up to 45%, preferably 5-20%, of a comonomer. The biologically active substances are substances such as enzymes, Activators, inhibitors, antigens or antibodies, other plasma proteins, blood group substances, phytemagglutinins, antibiotics, vitamins or hormones, Peptides or amino acids or synthetically produced effectors.

The polymeric carrier and the biologically active substance are either directly or through a Side chain (spacer) linked covalently to one another

Binding the biologically active compounds via a side chain (spacer) is advantageous if the Molecular weights of their affine Psatner are very different, or when in use in iron biospecific process take part in very high molecular weight proteins or proteins composed of several subunits. Spacers are anchored to the polymer matrix Side chains of a certain length, which are polymerized by attachment or incorporation (binding of bi- or polyfunctional compounds - e.g. B. a tripeptide - to the matrix) or by incorporation of functional groups and gradual extension jo on these can be obtained.

The invention also relates to processes for the covalent connection of the biologically active substances with the carrier.

A number of well-known reactions are available for this purpose.

They initially relate to the process for producing the polyvinyl glycol copolymers. You are through it characterized in that vinylene carbonate with comonomers, especially those of the general Formulas:

HjC = C

COOR, R.

Ο —CO —CH, R,

50

H, C =

Cl

H ₂ C =

CH = CH ₂

H ₂ C = C

(OCH ₂ -CHi) _n -O-CH =

H ₂ C = C CH ₂

N (CH ₂ ),

OC CH ₂

R.

H ₂ C =

CH ₂

Ri

H ₂ C = C

O —CH CH ₂

H ₂ C = C

CH-CH ₂

I I ο ο

Il ο

in which Ri is hydrogen, methyl, ethyl, R _{3 is} methyl, eth;, i propyl and η is an integer between 1 and 4 mean, is polymerized.

The polyvinylene carbonate is polymerized with up to 45% comonomers, preferably mix 5-20%. The binding of the effector to the polymeric carrier

M) can - taking into account the ability of the Polyvinyl carbonate for hydrolysis and the tendency to part of the bound in the polymer To get hydroxyl groups - done in several ways.

h) So you can:

(A) reacting a copolymer of vinyl carbonate with a biologically active substance and thereafter

convert the cyclocarbonate groups still present into hydroxyl groups or
(B) converting the cyclocarbonate groups of a copolymer of vinylene carbonate into hydroxyl groups and

(A) present in the copolymer or introduced into this electrophilic groups with a convert biologically active substance or

(b) these hydroxy groups

(1) either first with an electrophilic group-containing compound and then with a biologically active substance

(2) or directly with a biologically active substance carrying electrophilic groups realize.

The manufacture of those bound to the carrier

possible because an electrophilic group either in the biologically active substance or in the Polyvinyl glycol copolymer is introduced and through this the biologically active substance with the polyvinyl glycol copolymers is implemented.

The introduction of the electrophilic groups into the carrier matrix takes place either by copolymerization of vinylene carbonate with comonomers containing electrophilic groups or by reacting the Carrier matrix with activating, low molecular weight compounds bearing electrophilic groups.

In general, the chemical coupling of a reactant to the carrier matrix is easy when on the nucleophilic functions on one side and electrophilic functions on the other. As a reactive nucleophile Functions are mainly amino, sulfhydryl and hydroxyl groups into consideration, usually either are already contained in the matrix or in the reaction partner. Electrophilic functions have to be done first to be introduced. For this purpose, carboxyl groups in acid halides, acid azides, acid anhydrides, imidazolides transferred or activated directly with carbodiimides. As reactive electrophilic groups are also isocyanate, isothiocyanate, diazonium groups or cyclic imidocarbonate esters are used. The introduction of more reactive is particularly favorable Groups with the help of copolymerization.

By polymerizing monomers with epoxy groups, e.g. B. with Epoxyalkylvinyläthern in the > Polyvinylene carbonate and due to the very large differences in the saponification rate between cyclic carbonate and epoxy groups. a polyvinyl glycol matrix with reactive Epoxy groups which can be reacted with nucleophilic functions ι ·). Such is the direct bond a biologically active substance and / or the introduction (extension) of a side chain (spacer) is possible.

Another advantage is therefore in the use

of copolymers also the possibility of direct

Ii Introduction (polymerisation) of electrophiles Functions that allow implementation using known methods.

By copolymerizing vinylene carbonate with

JL | U ** 1. Λ sif> wla * li * ^ r! SChfc! ' ^v0n ^ ^or Λ / ArcjktfitnfT A & r PnIw.

meren you get a matrix with -OH- and - COOH functions. The carboxyl groups can be activated with the help of carbodiimides and then enter into an amide bond with amino groups of the effector. Carboxyl groups also leave the

λ> Formation of amide bonds with the help of Woodward introduced (soxazolium salts to.

Another method for coupling biologically active compounds to those containing carboxyl groups Polymers is made with the help of N-ethoxycarbonyl-2-eth-

so carried out oxy-i ^ -dihydroquinoline (EEDQ).

The carboxyl groups can be esterified by known methods, then the ester into the hydrazide transferred and finally the resulting azide with the amino group of the effector protein with the

r. Polyvinyl glycol matrix can be linked.

Has the comonomer polymerized into the matrix through subsequent reactions freely available amino groups, so is with containing arylamino groups Vinyl sulfone derivatives or sulfuric acid esters of

jo 0-Hydroxyäthylsulfonen the introduction of arylamino groups possible, the arylamino groups then diazotized by known methods and then can be associated with corresponding reactive groups of an effector, e.g. B.

HH
-C —C -
OH OH

HHHH

-C - C - C - C

OH H O H

CH ₂
HIGH

CH ₂ - NH— (CH ₂ ) ,, —- NH —CH ₂ -

-NH ₂

In this way, a “lengthening” of a spacer can also be carried out.

Another way of binding proteins via the amino groups is with the help of glutaraldehyde.

It is easy to link the effector to the matrix after activation of the hydroxyl or Amino groups of the Polyvinyiengiykol copolymers by means of cyano halides, advantageously with cyanogen bromide and a subsequent reaction of the amino groups

containing biologically active effectors via these activated groups.

Another possibility for linking the effector with a polyvinyl glycol or polyvinyl-Iene glycol copolymer matrix consists in the acylation of the hydroxyl groups with bromoacetyibromide, followed by the alkylation of the amino groups of the effector.

Similar reaction courses are, for example, through

Implementation of the hydroxyl groups of the carrier with reactive triazines to achieve part of the reactive groups of the triazine with the polyvinyl glycol compound, another with amino groups of the Effector reacts.

Diazolizable aromatic amines which have a further reactive group with the hydroxyl groups of the Wearer can connect on the one hand, deaden the coupling on the other hand enabled, activated amino acids, for example the tyrosine or histidine residues of the protein effector.

Vinylsulfone derivatives containing arylamino groups and sulfuric acid half esters of / f-Hydroxyäthylsulfonen can with the hydroxyl groups of the carrier for Reaction to be brought. They enable the binding of the effector as described above Diazotization reaction.

Particularly stable ether compounds are formed when the hydroxyl groups of the polyvinyl glycol copolymer are reacted with non-ion-forming epoxides that contain at least 2 reactive groups, such as the epihalohydrins or the polyepoxides, for example epichlorohydrin or bisepoxides.

Another type of modification is the polymerisation of comonomers into the polyvinylene carbonate chain (such as vinylpyrrolidone) with subsequent

ι «Bender partial implementation of the cyclocarbonate rings of the polyvinylene carbonate with an amine, e.g. B. with hexamethylenediamine, to a substituted via a urethane bond with a spacer or effector Polyvinylene carbonate copolymer. The remaining

Ii cyclocarbonate groups are then saponified to hydroxyl groups:

-CH
ι -CH ₂ - γ / "CH- CH"
ι ι ^J + P ft -NH ₂ H ₂ -CH -CH ₂ - γ / "CH-
I. ^L. CH
I. - -CH
j - ( I.
N O I I
O O N O I.
O I.
O I.
OH :H - H ₂ / \ H ₂ \ / H ₂ / \ H ₂ \ / / O L. C. L. C. H: Il Il carbonate O O C = O n- I pole vvinvler NH R.

-CH-CH ₂ -

Saponification

CH-CH "

I I

OH OH

CH
I. -CH I.
OH O NH
I. I.
R.

In addition to the processes exemplified here for producing the covalent connection between the carrier matrix and the protein effector or v, other effectors, there are other methods that lead to the reaction of the hydroxyl groups or certain groups of the copolymer with an effector and thereby produce the covalent connection between the two ; so the well-known implementation with complex-forming metal compounds, z. B. titanium compounds. All of these methods are known to the person skilled in the art. When binding low molecular weight biologically active compounds, it is often advantageous not to activate the carrier but rather the effector. ■>■>

In principle, all methods can be used which are known in macromolecular chemistry for the modification of synthetic or natural macromolecular compounds.

The polyvinyl glycol polymers are characterized by w) in addition to chemical and thermal stability through advantageous processing properties and thus lead to a superiority over the carrier matrices on the which have been found to be optimal up to now Based on natural carbohydrates. You are e.g. B. in t> 5 Form of fibers, threads, foils or spherical particles can be produced, so that the most suitable one, depending on the intended use of the effector to be attached to it Shape can be chosen. These copolymers are preferably in the form of finely divided powders applied with a high specific surface.

Due to the controllable size for the binding of the effectors or the spacers accessible surface shows a further advantage of the polyvinyl glycol copolymers over the known carrier materials.

The biologically active compounds according to the invention are suitable for most of the application methods which have hitherto become known for other effectors bound to hydrophilic, water-insoluble carriers are. Thus, enzymes can be made water-insoluble. The enzymes become insoluble increasingly for the determination of substrates in automatic analyzers and as so-called enzyme electrodes used Because of the increased stability, a number of carrier-bound enzymes are suitable for carrying out technical enzymatic conversions.

Carrier-bound, biologically active substances have found wide application in affinity chromatography because of their property as specific adsorbents. With the help of carrier-bound natural or synthetic enzyme inhibitors succeed in the high purification of enzymes, while the Enzymes as effectors, in particular for extraction

i |

natural enzyme inhibitors from crude extracts have been found to be extremely suitable. Carrier-bound Water-insoluble antigens are used to isolate the associated antibodies raised in this way free from other serum components and free from other antigens. Affinity chromatography can also be non-precipitable Antibodies as well as those which cannot be precipitated due to their low concentration in the serum are isolated and can also be determined quantitatively.

Examples Production of vinylene carbonate copolymers

The '' creation of the copolymers (CP) The vinylene carbonate used was dried over sodium borohydride (100 parts by weight Vinylene carbonate on 2 parts by weight of NaBH *) boiled under reflux at a pressure of 33 mm, then at

iigc, mil rvascmg-vjias-

2.) C'opolymcrisat vinyl glycol / yinyl glycidyl ether

A mixture of 7 parts by weight of vinylene carbonate, 3 parts by weight of vinyl glycidyl ether and 0.1 part by weight Azobisisobutyronitrile is as described under I.) a) in flat aluminum tubes under N2 for 3 days at 50 ° warmed up. The plastic sheets obtained are comminuted and demonomerized as described under 1.) a). ground to a grain size <0.1 mm, in 500 parts by weight of ice-cold, 0.5 η NaOH with a high-speed stirrer suspended for 30 minutes, then immediately centrifuged the saponified polymer, slurried in ice water, while cooling with ice 2 η H2SO4 neutralized to pH 7, suction filtered, washed out with ice water and freeze-dried. Yield: 3.9 parts by weight of CP vinyl glycol (vinyl glycidyl ether containing 1.3 milliequivalents of oxirane groups / g (determined according to: »Practical course in macro-

ClIlC JVLII VGfi υΓαίϊΠ,

Ring-filled silver jacket column is distilled and used as soon as possible for the copolymerization. The comonomers used were also used previously purified by distillation.

1.) Copolymer vinyl glycol / vinyl alcohol made of saponified vinylene carbonate / vinyl acetate copolymer

a) 0.05 parts by weight of azobisisobutyronitrile are dissolved under nitrogen in 9 parts by weight of vinylene carbonate and 1 part by weight of vinyl acetate. The monomer mixture is pressed under nitrogen into a flattened (4 mm thick, 60 mm wide, 150 mm long) aluminum tube filled with screw cap (total filling volume approx. 35 ml), Sealed under N2 and hung in a 50 ° warm water bath for 48 h. It becomes hard, brittle Obtain plastic plates that are pre-crushed in a hammer mill. Then will the granules are heated at 120 ° for 5 hours at 1-2 mm pressure in order to separate off the unpolymerized residual monomers. The demonomerized granulate is in a mill -weiter on one grain size <0.1 mm ground, registered in 500 parts by weight of 0.5 N sodium hydroxide solution, at 20 ° with a high-speed stirrer mixed for one hour, saponifying the carbonate and acetyl groups will. The water-insoluble CP vinyl glycol / vinyl alcohol is suctioned off, well with water Washed free of inorganic salts and freeze-dried

Yield: 4.7 parts by weight of CP vinyl glycol / vinyl alcohol with a BET specific surface area of 34 m ² g- '.

b) As described under 1.) a), except that 3 parts by weight per 7 parts by weight of vinylene carbonate Vinyl acetate can be used.

Yield: 4.1 parts by weight of CP vinyl glycol / vinyl alcohol with a specific surface area of freeze-dried polymer powder of

c) Comparative experiment as described under I.) a), only that 10 parts by weight of vinylene carbonate and no comonomer are used. Yield: 4.7 parts by weight of homopolymer vinyl glycol with a specific surface area of freeze-dried polymer powder of 20.5

MiOiciCüiSrCn CrgämSCitcM ^ iiCiiiiC «, > j.

D. Cherdron, H., Kern, W, Müthig Verlag. Heidelberg 1966).

3.) Amino-substituted copolymers. Carrier material

3.9 parts by weight of CP vinyl glycol / vinyl glycidyl ether (from Example 2.)) are suspended in 100 parts by weight of H2O with an Ultra-Turrax-P stirrer and with 10 ml 30% ammonia added. With a little one

a magnetic stirrer is then used for 10 hours at 50 ° stirred, finally suctioned off and washed well with water and freeze-dried. Yield: 3.5 parts by weight of copolymer carrier material containing 0.9 m equivalent amino groups

ji per gram, in the form of 3-amino-2-hydroxypropyl groups (from the reaction of ammonia with Glycidyl groups formed).

4.) CP vinyl glycol / aryl acid

jo 9 parts by weight of vinylene carbonate, 1 part by weight Acrylic acid ethyl ester and 0.05 part by weight of azobisisobutyronitrile are under N2 as in BeispiM 1.) a) Dolvmerized, crushed, demonomerized and cut to a grain size <0.1 mm ground, saponified, > soaks, washed out and freeze-dried.

Yield: 5.0 parts by weight of hydrophilic polymer powder containing 4.1 mole equivalents of carboxyl groups per gram of carrier with a BET specific surface area of 27.2 m ² g- '.

5.) ü) -Aminohexyl groups-substituted Poly vinyl glycol

5 parts by weight of CP vinyl glycol / acrylic acid from Example 4) are mixed with the Ultra-Tuirax in 100 ml of H ₂ O suspended, cooled to 5 °, with 2.5 g of N-Cyclo hexyl-N'-iN-methylmorpholinoJ-ethyiycarbodiimido-

p-toluene sulfonate added, 30 minutes at pH 5 and 5 ° stirred, filtered off and washed quickly with ice water.

The activated carrier is then immediately converted into a

bo set to pH 7.5 with 2η HCl and cooled to 5 ° Solution of 5 g of hexamethylenediamine suspended in 100 ml of water and a further 24 hours under this Conditions stirred with a magnetic stirrer, suctioned off, washed well with water and freeze- dries

Yield: 4.5 parts by weight of ω-aminohexyl group-substituted polyvinyl glycol with 0.7 milliequivalents of amino groups per gram of carrier.

6.) Reaction of CP vinyl glycol / vinyl alcohol from Example 1.) a) with a BET specific surface area of 34 m ² g - '

») With epichlorohydrin

50 g of a CP vinyl glycol / vinyl alcohol, prepared according to Example 1.) a) are 2 η in 1 liter Suspended NaOH, treated with 250 ml of epichlorohydrin and stirred for 2 hours at 55-60 °. Of the The pH of the suspension drops to 10-11 after a short time. By adding NaOH, 1 This pH value was maintained for an hour. After a reaction time of 2 hours, the solid is filtered off with suction, with Water, acetone and finally washed again with water.

b) with hexamethylenediamine

50 g of hexamethylenediamine are dissolved in 1.5 liters of water, and HCl is added to pH 10. To the The solution is the CP vinyl glycol / vinyl alcohol activated according to 6.) a) given and stirred at 50-55 ° for 6 hours. The product is then filtered off with suction and free from hexamethylenediamine with water washed.

c) with i-aminobenzene ^ - ^ - hydroxyethylsulfonic acid ester

That for example! 6.) b) The product obtained is treated with 50 g of i-aminobenzene-4-ß-hydroxyethylsulfonic acid ester Stirred for one hour at 55 "-ind pH 10. Then the solid filtered off, washed with water, acetone and again with water.

d) diazotization

10 g of the product obtained under Example 6.) c) are washed on a suction filter with 200 ml of η HCl and then suspended in 300 ml of 0.5 η HCl. The suspension is mixed with 0.1 η NaNO ₂ solution at 0-4 °, while stirring, until a slight excess of nitrite can be observed during the diazotization with KJ starch paper. After 10 minutes, a suction filter is filtered off and the residue is washed with ice water and then with 0.15 M sodium phosphate buffer pH 7.5 at 0-4 °.

e) Covalent bond with protein

0.8 g of albumin are dissolved in 350 ml of phosphate buffer pH 7.5, cooled to 4 ° and the product prepared under 6.) d) is added. The suspension is stirred for 20 hours at 4 °, then filtered off and the solid is washed with 1 M NaCl and phosphate-buffered saline (PBS) (aqueous 03% NaCl solution containing 1 / 15M Na ₂ HPO ₄ -KH ₂ PO ₄ buffer of pH 7.2).

The filtrate and wash liquor are examined for albumin using the radial immunodiffusion method 60 mg of albumin are bound to 1 g of the carrier produced in this way.

7.) Implementation of CP vinyl glycol / vinyl alcohol from Example 1.) b) with a BET specific surface area of 62 ^ m ² g- '

After reactions similar to those described under 6.) a) to e), 1 g of activated carrier 80 mg Albumin can be bound quantitatively.

8.) Reaction of homopolymer vinyl glycol from Example 1.) c) with a BET specific surface area of 20.5 m ² g- ¹

After analogous reactions as described under 6.) a) to e), 45 mg of activated carriers can be obtained from 1 g Albumin can be bound quantitatively.

9.) w- _{Aminohexyl groups-substituted, n} vinyl glycol / vinyl pyrrolidone copolymer

In 9 parts by weight of vinylene carbonate and 1 part by weight of vinylpyrrolidone there are 0.05 parts by weight of azobisisobutyrolonitrile Dissolved under nitrogen and polymerized and worked up analogously to Example 1.) a).

ι ") After demonomerization, the granules become a 12 weight percent dimethylform

amide solution, and with a pressure of 15Atü this solution is sprayed into a falling methanol bath. A fine-fibrous precipitate is obtained

-'Ii Vinyiencarbonat / Vinyipyrroiidon-Copoiymerisat, the is suctioned off, washed with methanol and resuspended in 200 weight stiffeners of methanol. It will 6 parts by weight of hexamethylenediamine, dissolved in 50 parts by weight of methanol, are added, suspension 2

Stirred for 2ϊ days at room temperature, suction filtered and with Washed out methanol. The washed filter residue is now in a solution of 10 parts by weight Sodium methylate suspended in 300 parts by weight of 96% methanol for 4 days at room temperature, with

washed out in methanol and then very intensively with water and freeze-dried

Yield: 5.0 parts by weight of one substituted with ω-aminohexyl groups via urethane bonds Vinyl glycol / vinyl pyrrolidone copolymer containing

r> tend 1.6 meq. NH ₂ ribs / g carrier.

10.) Binding of IgG to ω-aminohexyl group-substituted copolymer from Example 9.) with the aid of succinic anhydride and _4N water-soluble carbodiimide

5 parts by weight of ω-aminohexyl-substituted carrier prepared according to Example 9.) are at 10 ° and nH fi mi »? S Opwirhtstpilpn Rprns "pin" änrpanhyrlriH Hip are suspended in 200 parts by weight of water, 4

Succinylated for 4 hours. The pH value is 2 η NaOH regulated. After washing the solid with water, the product is 1.25 parts by weight N-Cyclohexyi-N'-tN-methymorpholinoJ-ethylJ-carbodiimide-p-toluene-sulfonate Stirred at pH 5 and 5 ° for 30 minutes, filtered off and washed quickly with ice water.

0.5 parts by weight of IgG are dissolved in 150 parts by weight of phosphate buffer pH 7.5 and mixed with the activated one The carrier is stirred for 24 hours at 4 °. After filtration, the product is washed with 1 M sodium chloride and with PBS. 75 mg IgG are covalently bound to 1 g of the carrier.

11.) Copolymer vinyl glycol Diethylene glycol divinyl ether

In 9 parts by weight of vinylene carbonate and 1 part by weight of diethylene glycol divinyl ether, 0.1 part by weight of azobisisobutyrolnitrile is dissolved under nitrogen and _{heated as described under 1.) a) in flat aluminum tubes under N 2 for} 3 days at 50 ° and polymerized. After work-up and saponification as under 1.) a ) described 3.5 parts by weight of CP vinyl glycol / diethylene glycol divinyl ether are obtained.

ί5

1 2.) Copolymer vinyl glycol / N-acryloylaminoacetaldehyde dimethylacetaJ

a) In 9 parts by weight of vinylene carbonate and 1 part by weight of N-acyloylaminoacetaldehyde dimethyl acetal, 0.05 parts by weight of azolisisobutyronyltrile are dissolved under nitrogen.
10 parts by weight of N-acryloylaminoacetaldehyde di-

methyl acetal / vinyl glycol copolymers were stirred in 100 parts by weight IN HCl for 4-5 hours The activated carrier was washed with water and phosphate buffer pH 7.5,
b) 0.5 g of albumin was dissolved in 200 ml of PBS and stirred for 14 hours at 4 ° with the product prepared under a). After filtration, the carrier-bound protein was washed with 1 M sodium chloride and with PBS.

1 g of the carrier produced in this way binds 40 mg of albumin.

130 208/209

Claims (5)

Patent claims: 1. Compound of a copolymer of Vinyl glycol and a biologically active substance chemically bound to it, in which only a part the active groups of the vinyl glycol units of the polymer to the biologically active substance is bound and the remainder is present as free hydroxyl groups 2. Compound according to claim 1, characterized in that the copolymer at least Contains 55% monomer units of vinyl glycol and a maximum of 45% monomer units at least one compound of the general formulas: R, 10 15th H ₂ C = C 20th COORj H ₂ C = H ₂ C = Ο —CO —CHj R ₁ Cl R. JO J5 H ₂ C = C HjC = H ₁ C = C (OCH, -CHj) ₁₁ -O-CH = CH ₃ N (CW ₁ ) ,. i! (K CH, H ₂ C = C (CHj), - CH CH ₂ Ri H ₂ C = C O —CH CH ₂ H ₂ C = C CH-CH ₂ I I ο ο in which Ri is hydrogen, methyl, ethyl, R _{3 is} methyl, ethyl, propyl and η is an integer between 1 and 4. 3. Compound according to claim I, characterized in that the biologically active substance an enzyme, activator, inhibitor, antigen or antibody, a plasma protein, a blood group substance, a phythemagglutinin, an antibiotic, Vitamin or hormone, a peptide or an amino acid, a natural or synthetically produced effector 4. Process for the preparation of a compound according to claim I, characterized in that man (A) reacting a copolymer of vinyl carbonate with a biologically active substance and thereafter the remaining cyclocarbonate groups in hydroxyl groups or (B) the cyclocarbonate groups of a copolymer of vinylene carbonate are converted into hydroxyl groups and (A) present in the Copolymensat or introduced into this electrophilic groups with a converts biologically active substance or (b) these hydroxy groups (1) either first with an electrophilic group-containing compound and then with a biologically active substance (2) or directly with an electrophilic group (outstanding biologically active Substance converts. 5. Use of a compound according to claim 1 for affinity chromatography to carry out of immune reactions or to carry out enzyme reactions.